为构建携带水稻瘤矮病毒(Rice gall dwarf virus,RGDV)主要内层衣壳蛋白S3基因和外层衣壳蛋白S8基因的重组杆状病毒,将目的基因(S3和S8)分别亚克隆到杆状病毒转移载体pFastBacDual多角体启动子(PH)和p10启动子的下游.经酶切和确证性序...为构建携带水稻瘤矮病毒(Rice gall dwarf virus,RGDV)主要内层衣壳蛋白S3基因和外层衣壳蛋白S8基因的重组杆状病毒,将目的基因(S3和S8)分别亚克隆到杆状病毒转移载体pFastBacDual多角体启动子(PH)和p10启动子的下游.经酶切和确证性序列测定,将其转化到DH10Bac感受态细胞中,获得重组杆粒rbpFBDS3-S8,采用脂质体转染法,将rbpF-BDS3-S8转染草地贪夜蛾(Spodoptera frugiperda)Sf9细胞包装病毒,PCR筛选鉴定重组病毒.结果表明:Sf9昆虫细胞被侵染72 h后,倒置显微镜下观察到细胞增大,培养液和细胞内出现颗粒状物质,部分细胞破裂甚至裂解,说明S3和S8基因已整合到重组杆状病毒基因组中,这为开展RGDV主要结构蛋白在昆虫细胞中的表达及其功能研究奠定了基础.展开更多
To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8...To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.展开更多
Rice gall dwarf virus(RGDV)is an important rice pathogen in China and Southeast Asia.However,little is known about the molecular mechanisms of RGDV interactions with plant cells.Here,we have identi-fied an RGDV protei...Rice gall dwarf virus(RGDV)is an important rice pathogen in China and Southeast Asia.However,little is known about the molecular mechanisms of RGDV interactions with plant cells.Here,we have identi-fied an RGDV protein,Pns11,which acts as a suppressor of RNA silencing in coinfiltration assays with the reporter,green fluorescent protein(GFP)in transgenic Nicotiana benthamiana line 16c carrying GFP.Pns11 suppressed local and systemic silencing induced by sense RNA.The spread of mobile RNA si-lencing signals was blocked or inactivated by Pns11.Expression of Pns11 also enhanced Potato virus X pathogenicity in N.benthamiana.This suppressor could reduce,but not eliminate,siRNA in the local and systemic RNA silencing suppression assays,suggesting that Pns11 functions by interfering with initial stages of RNA silencing.展开更多
基金supported by the National Science Foundation of China (30970135)The Key Project of Genetically Modified Organisms Breeding(2009ZX08009-044B)+1 种基金the Natural Science Foundation of Fujian Province of China (No.2006J0065)the Public-interest Scientific Institution Basal Research Fund of Fujian Province (2009R10029-3)
文摘To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.
基金the National Natural Science Foundation of China(Grant Nos.30370929 and 30671358)
文摘Rice gall dwarf virus(RGDV)is an important rice pathogen in China and Southeast Asia.However,little is known about the molecular mechanisms of RGDV interactions with plant cells.Here,we have identi-fied an RGDV protein,Pns11,which acts as a suppressor of RNA silencing in coinfiltration assays with the reporter,green fluorescent protein(GFP)in transgenic Nicotiana benthamiana line 16c carrying GFP.Pns11 suppressed local and systemic silencing induced by sense RNA.The spread of mobile RNA si-lencing signals was blocked or inactivated by Pns11.Expression of Pns11 also enhanced Potato virus X pathogenicity in N.benthamiana.This suppressor could reduce,but not eliminate,siRNA in the local and systemic RNA silencing suppression assays,suggesting that Pns11 functions by interfering with initial stages of RNA silencing.
